Multivesicular liposomes with controlled release of encapsulated biologically active substances

ABSTRACT

A multivesicular liposome composition containing at least one acid other than a hydrohalic acid and at least one biologically active substance, the vesicles having defined size distribution, adjustable average size, internal chamber size and number, provides a controlled release rate of the biologically active substance from the composition. A process for making the composition features addition of a non-hydrohalic acid effective to sustain and control the rate of release of an encapsulated biologically active substance from the vesicles at therapeutic levels in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/898,017, filed Jul. 21, 1997, now abandoned, which is a continuationof U.S. patent application Ser. No. 08/473,013, filed Jun. 6, 1995, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 08/153,657, filed Nov. 16, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to compositions of multivesicular liposomes usefulas a drug delivery system and processes for their manufacture.

2. Description of Related Art

Optimal treatment with many drugs requires maintenance of a drug levelfor an extended period of time. For example, optimal anti-cancertreatment with cell cycle-specific antimetabolites requires maintenanceof a cytotoxic drug level for a prolonged period of time. The half-lifeof many drugs after an intravenous (IV), subcutaneous (SC),intraperitoneal (IP), intraarterial (IA), intramuscular (IM),intrathecal (IT), or epidural dose is very short, being in the range ofa fraction of an hour to a few hours. Cytarabine is a highlyschedule-dependent anti-cancer drug. Because this drug kills cells onlywhen they are making DNA, prolonged exposure at therapeuticconcentration of the drug is required for optimal cell kill. To achieveoptimal cancer cell kill with a cell cycle phase-specific drug likecytarabine, two major requirements need to be met: irreversible harm tothe host; and second, the tumor must be exposed for a sufficient lengthof time so that all or most of the cancer cells have attempted tosynthesize DNA in the presence of cytarabine.

An example of another class of drugs that are schedule-dependent is theclass of aminoglycoside antibiotics. For instance, amikacin is anaminoglycoside antibiotic that has clinically significant activityagainst strains of both gram negative and gram positive bacteria, buthas a serum half-life of about two to three hours. Yet in currentpractice, the drug is normally administered by intravenous orintramuscular routes once or twice a day. The most commonly usedclinical dose is 15 mg/Kg/day, which is equivalent to a maximumrecommended daily dose of 1 g per day.

For infections such as those confined to a local region of soft tissueor bone, an implantable drug depot with sustained release propertieswould be advantageous, both to increase local levels of the drug in theaffected tissue and to reduce or avoid the systemic toxicity of the freedrug.

Thus, new and better methods for sustained release delivery of drugs inthe treatment of disease are needed. The present invention meets thisneed by providing compositions of multivesicular liposomes useful as asustained release drug delivery system and a process for theirmanufacture.

Multivesicular liposomes (MVL), first reported by Kim, et al. (Biochim,Biophys. Acta, 728:339-348, 1983), are uniquely different from otherlipid-based drug delivery systems such as unilamellar (Huang,Biochemistry, 8:334-352, 1969; Kim, et al., Biochim. Biophys. Acta,646:1-10, 1981) and multilamellar (Bangham, et al., J Mol. Bio.,13:238-252, 1965) liposomes. The main structural difference is that incontrast to unilamellar liposomes (also known as unilamellar vesicles,or "ULV"), multivesicular liposomes (MVL) contain multiple aqueouschambers per particle. In contrast to multilamellar liposomes (alsoknown as multilamellar vesicles or "MLV"), the multiple aqueous chambersin multivesicular liposomes are non-concentric. The structuraldifferences between unilamellar, multilamellar, and multivesicularliposomes are illustrated in FIG. 1.

Because of the similarity in acronyms, multivesicular liposomes (MVL)are frequently confused with multilamellar liposomes (MLV).Nevertheless, the two entities are entirely distinct from each other.The structural and functional characteristics of MVL are not directlypredictable from current knowledge of ULV and MLV. As described in thebook edited by Jean R. Philippot and Francis Schuber (Liposomes as Toolsin Basic Research and Industry, CRC press, Boca Raton, Fla.,1995, page19), MVL are bounded by an external bilayer membrane shell, but have avery distinctive internal morphology, which may arise as a result of thespecial method employed in the manufacture. Topologically,multivesicular liposomes (MVL) are defined as liposomes containingmultiple non-concentric chambers within each liposome particle,resembling a "foam-like" matrix; whereas multilamellar vesicles (MLV)contain multiple concentric chambers within each liposome particle,resembling the "layers of an onion".

The presence of internal membranes distributed as a network throughoutMVL may serve to confer increased mechanical strength to the vesicle,while still maintaining a high volume:lipid ratio compared with MLV. Themultivesicular nature of MVL also indicates that, unlike for ULV, asingle breach in the external membrane of a MVL will not result in totalrelease of the internal aqueous contents. Thus, both structurally andfunctionally the MVL are unusual, novel and distinct from all othertypes of liposomes. As a result, the functional properties of MVL arenot predictable based on the prior art related to conventional liposomessuch as ULV and MLV.

The prior art describes a number of techniques for producing ULV and MLV(for example, U.S. Pat. Nos. 4,522,803 to Lenk; 4,310,506 toBaldeschwieler; 4,235,871 to Papahadjopoulos; 4,224,179 to Schneider;4,078,052 to Papahadjopoulos; 4,394,372 to Taylor; 4,308,166 toMarchetti; 4,485,054 to Mezei; and 4,508,703 to Redziniak). The priorart also describes methods for producing MVL (Kim, et al., Biochim.Biophys. Acta, 728:339-348, 1983). For a comprehensive review of variousmethods of ULV and MLV preparation, refer to Szoka, et al., Ann. Rev.Biophys. Bioeng.,9:465-508, 1980.

In the method of Kim, et al. (Biochim. Biophys. Acta, 728:339-348,1983), the pharmaceutical utility of MVL encapsulating small therapeuticmolecules, such as cytosine arabinoside or cytarabine, is limited.Subsequent studies (Kim, et al., Cancer Treat. Rep., 71:705-711, 1987)showed that the release rate of encapsulated molecules into biologicalfluids can be modulated by encapsulating in the presence of ahydrochloride.

Heretofore, control of the release rate of a biologically activesubstance from multivesicular liposomes could only be achieved by use ofhydrohalides. For a drug-delivery system, it is highly advantageous tobe capable of controlling the release rate for encapsulated substancesthrough release rate modifying agents used during manufacture of theliposomes, and to have a wide choice of these release-rate modifyingagents.

Accordingly, it is an object of the present invention to provide acontrolled release depot preparation of multivesicular liposomes whichprovides a sustained exposure of a biologically active substance at atherapeutic concentration.

It is a further object of the present invention to provide a method ofpreparing such depot preparations.

Other and further objects, features, and advantages of the invention areinherent therein and appear throughout the specification and claims.

SUMMARY OF THE INVENTION

The compositions of the present invention comprise multivesicularliposomes (MVL), i.e. lipid vesicles with multiple internal aqueouschambers formed by non-concentric layers, and having internal membranesdistributed as a network throughout the MVL, wherein the chamberscontain one or more non-hydrohalic acids effective in controlling therelease rate of the encapsulated biologically active substance. Thepresent invention also provides methods of making such compositions.

The present multivesicular liposome compositions have high encapsulationefficiency, controlled release rate of the encapsulated substance, welldefined, reproducible size distribution, adjustable average size thatcan be easily increased or decreased, and adjustable internal chambersize and number.

The process for producing these MVL compositions comprises (a) formingan emulsion from a lipid component comprising at least one organicsolvent, at least one amphipathic lipid, at least one neutral lipid, andan immiscible first aqueous component comprising at least onebiologically active substance and, in the presence of at least onenon-hydrohalic acid, (b) mixing the emulsion with a second aqueouscomponent to form solvent spherules, (c) removing the organic solventfrom the solvent spherules to form multivesicular liposomes. Accordingto the present invention, addition of one or more non-hydrohalic acidsis effective in controlling the release rate of the encapsulatedbiologically active substance into biological fluids and in vivo.

DESCRIPTION OF THE DRAWING

FIG. 1 shows illustrations comparing the internal structures of aunilamellar liposome, a multilamellar liposome, and a multivesicularliposome.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "multivesicular liposomes" (MVL) as used throughout thespecification and claims means man-made, microscopic lipid-vesiclesenclosing multiple non-concentric aqueous chambers formed by internalmembranes distributed as a network throughout the MVL. In contrast,unilamellar vesicles (ULV) have a single aqueous chamber; andmultilamellar liposomes (MLV) have multiple "onion-skin" type ofconcentric membranes, in between which are concentric aqueouscompartments.

The term "solvent spherule" as used throughout the specification andclaims means a microscopic spheroid droplet of organic solvent, withinwhich are suspended multiple smaller droplets of aqueous solution.

The term "neutral lipid" means oils or fats that have nomembrane-forming capability by themselves and lack a hydrophilic "head"group.

The term "amphipathic lipids" means those molecules that have ahydrophilic "head" group and hydrophobic "tail" group and havemembrane-forming capability

The term "zwitterionic lipid" means an amphipathic lipid with a netcharge of zero at pH 7.4.

The term "anionic lipid" means an amphipathic lipid with a net negativecharge at pH 7.4.

The term "cationic lipid" means an amphipathic lipid with a net positivecharge at pH 7.4

The term "hydrohalic acid" means hydrofluoric acid, hydrochloric acid,hydrobromic acid, hydroiodic acid, or a combination thereof.

The term "biologically active substance" as used herein means a chemicalcompound, other than any acid used as a release-rate modifying agentaccording to the present invention, that is known in the art as havingutility for modulating biological processes so as to achieve a desiredeffect in modulation or treatment of an undesired existing condition ina living being, such as a medical, agricultural or or cosmetic effect.Thus, biologically active substances are generally selected from thebroad categories of medicaments, radioisotopes, agricultural productsand cosmetics. Representative biologically active substances aredisclosed in Table 1 below.

Briefly, the preferred method of the invention for making MVL is asfollows. The first step is making a "water-in-oil" emulsion bydissolving amphipathic lipids containing at least one neutral lipid inone or more volatile organic solvents for the lipid component, adding tothe lipid component an immiscible first aqueous component and abiologically active substance to be encapsulated, and adding to eitheror both the lipid component and the first aqueous component, anon-hydrohalic acid effective in modulating the release rate of theencapsulated biologically active substances from the MVL. The mixture isthen emulsified, and then mixed with a second immiscible aqueouscomponent to form a second emulsion. The emulsions are formed eithermechanically, by ultrasonic energy, nozzle atomization, and the like, orby combinations thereof, to form solvent spherules suspended in thesecond aqueous component. The solvent spherules contain multiple aqueousdroplets with the substance to be encapsulated dissolved in them.

The organic solvent is removed from the spherules, generally byevaporation, for instance, by reduced pressure or by passing a stream ofgas over or through the suspension. When the solvent is completelyremoved, the spherules become MVL. Representative gases satisfactory foruse in evaporating the solvent include nitrogen, helium, argon, oxygen,hydrogen, carbon dioxide, or combinations thereof.

The non-hydrohalic acid present when the MVL is formed is effective incontrolling the rate of release of the encapsulated biologically activesubstance from the MVL into biological fluids and in vivo. The acidsinclude, but are not limited to, perchloric acid, nitric acid,glucuronic acid, citric acid, formic acid, acetic acid, trifluoroaceticacid, trichloroacetic acid, sulfuric acid, phosphoric acid, andcombinations thereof. The amount of the acid used is that effective toprovide a desired and controlled rate of release, which results intherapeutic levels of the encapsulated biologically active substancebeing released into a biological fluid or in vivo. For example, theconcentration of the non-hydrohalic acid in the lipid component or thefirst aqueous component to which it is added may be in the range of 0.1mM to about 0.5 M and preferably from about 10 mM to about 200 mM.

Many different types of volatile hydrophobic solvents such as ethers,hydrocarbons, halogenated hydrocarbons, or Freons may be used as thesolvent in the lipid component. For example, diethyl ether, isopropyland other ethers, chloroform, tetrahydrofuran, halogenated ethers,esters, and combinations thereof are satisfactory.

Various types of lipids can be used to make the multivesicularliposomes, and the only requirements regarding lipids for makingmultivesicular liposomes are that at least one amphipathic lipid and oneneutral lipid be included in the lipid component. The amphipathic lipidscan be zwitterionic, acidic or cationic lipids. Examples of zwitterionicamphipathic lipids are phosphatidylcholines, phosphatidylethanolamines,sphingomyelins etc. Examples of acidic amphipathic lipids arephosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, etc. Examples of cationic amphipathic lipids arediacyl trimethylammonium propanes, diacyl dimethylammonium propanes,stearylamine etc. Examples of neutral lipids include diglycerides, suchas diolein, dipalmitolein, and mixed caprylin-caprin; triglycerides,such as triolein, tripalmitolein, trilinolein, tricaprylin, andtrilaurin; and combinations thereof Additionally, cholesterol or plantsterols can be used to make multivesicular liposomes.

Many and varied biological substances and therapeutic agents can beincorporated by encapsulation within the MVL. The drugs that can beincorporated into the dispersion system as therapeutic agents includechemicals as well as biologics. The term "chemicals" encompassescompounds that are classically referred to as drugs, such as antitumoragents, anaesthetics, analgesics, antimicrobial agents, opiates,hormones etc. Of particular interest are amikacin, morphine,hydromorphone, cytarabine, methotrexate, 5-fluorouracil (5-FU),floxuridine (FUDR), bleomycin, 6-mercapto-purine, 6-thioguanine,fludarabine phosphate, vincristine, and vinblastine.

The term "biologics" encompasses nucleic acids (DNA and RNA), proteinsand peptides, and includes compounds such as cytokines, hormones(pituitary and hypophyseal hormones), growth factors, vaccines etc. Ofparticular interest are interleukin-2, insulin-like growth factor-1,interferons, insulin, heparin, leuprolide, granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), tumor necrosis factor, inhibin, tumor growth factoralpha and beta, Mullerian inhibitory substance, calcitonin, andhepatitis B vaccine.

The following TABLE 1 includes a list of classes of biologically activesubstances effective in humans that can be encapsulated in MVL in thepresence of a release-rate modifying non-hydrohalic acid of theinvention, and also includes biologically active substances effectivefor agricultural uses.

                  TABLE 1                                                         ______________________________________                                        Antianginas Antiarrhythmics                                                                              Antiasthmatic Agents                               Antibiotics Antidiabetics  Antifungals                                        Antihistamines                                                                            Antihypertensives                                                                            Antiparasitics                                     Antineoplastics                                                                           Antivirals     Cardiac Glycosides                                 Herbicides  Hormones       Immunomodulators                                   Monoclonal  Neurotransmitters                                                                            Nucleic Acids                                      Antibodies                                                                    Pesticides  Proteins       Radio Contrasts                                    Radionuclides                                                                             Sedatives and Analgesics                                                                     Steroids                                           Tranquilizers                                                                             Vaccines       Vasopressors                                       Anesthetics Peptides                                                          ______________________________________                                    

The term "therapeutically effective" as it pertains to the compositionsof the invention means that a biologically active substance present inthe first aqueous component within the vesicles is released in a mannersufficient to achieve a particular medical effect for which thetherapeutic agent is intended. Examples, without limitation, ofdesirable medical effects that can be attained are chemotherapy,antibiotic therapy, and regulation of metabolism. Exact dosages willvary depending upon such factors as the particular therapeutic agent anddesirable medical effect, as well as patient factors such as age, sex,general condition, and the like. Those of skill in the art can readilytake these factors into account and use them to establish effectivetherapeutic concentrations without resort to undue experimentation.

Generally, however, the dosage range appropriate for human use includesthe range of 0.1-6000 mg/sq m of body surface area. For someapplications, such as subcutaneous administration, the dose required maybe quite small, but for other applications, such as intraperitonealadministration, the dose desired to be used may be very large. Whiledoses outside the foregoing dose range may be given, this rangeencompasses the breadth of use for practically all the biologicallyactive substances.

The MVL may be administered for therapeutic applications by any desiredroute, for example, intramuscular, intraarticular, epidural,intrathecal, intraperitoneal, subcutaneous, intravenous, intralymphatic,oral and submucosal, and by implantation under many different kinds ofepithelia, including the bronchialar epithelia, the gastrointestinalepithelia, the urogenital epithelia, and various mucous membranes of thebody.

In addition, the MVL of the invention can be used to encapsulatecompounds useful in agricultural applications, such as fertilizers,pesticides, and the like. For use in agriculture, the MVL can be sprayedor spread onto an area of soil where plants will grow and theagriculturally effective compound contained in the vesicles will bereleased at a controlled rate by contact with rain and irrigationwaters. Alternatively the slow-releasing vesicles can be mixed intoirrigation waters to be applied to plants and crops. One skilled in theart will be able to select an effective amount of the compound useful inagricultural applications to accomplish the particular goal desired,such as the killing of pests, the nurture of plants, etc.

The following examples illustrate the manner in which the invention canbe practiced. It is understood, however, that the examples are for thepurpose of illustration and the invention is not to be regarded aslimited to any of the specific materials or conditions therein.

EXAMPLE 1

This example demonstrates that the release rate of a biologically activesubstance into an in vitro medium can be controlled by the use ofdifferent acids.

Step 1) In a clean glass cylinder (2.5 cm inner diameter×10.0 cmheight), 5 mL of a solution containing 46.5 μmoles of1,2-dioleoyl-sn-glycero-3-phosphocholine, 10.5 μmoles of1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 75 μmoles of cholesterol,9.0 μmoles of triolein in chloroform were placed (the lipid component.The lipids were purchased from Avanti Chemical Company (Alabaster,Ala.).

Step 2) Five mL of the first aqueous component, and cytarabine (20mg/mL) dissolved in 0.136 M of one of the acids to be tested was addedinto the above glass cylinder containing the lipid component. The acidstested as a release-rate modifying agent were: perchloric, nitric,formic, sulfuric, phosphoric, acetic, trichloroacetic, andtrifluoroacetic acids.

Step 3) For making the water-in-oil emulsion, a homogenizer(AutoHomoMixer, Model M, Tokushu Kika, Osaka, Japan) was used by mixingfor 8 minutes at a speed of 9000 rpm.

Step 4) For making the chloroform spherules suspended in water, 20 mL ofa solution containing 4 wt % glucose and 40 mM lysine was layered on topof the water-in-oil emulsion, and then mixed for 60 seconds at a speedof 4000 rpm to form the chloroform spherules.

Step 5) The chloroform spherule suspension in the glass cylinder waspoured into the bottom of a 1000 mL Erlenmeyer flask containing 30 mL ofwater, glucose (3.5 g/100 mL), and free-base lysine (40 mM). A stream ofnitrogen gas was passed at a flow-rate of 7 L/minute over the suspensionin the flask to evaporate chloroform over 20 minutes at 37° C. Sixty mLof normal saline (0.9% sodium chloride) was added to the flask. The MVLwere then isolated by centrifugation at 600 X g for 10 minutes. Thesupernatant was decanted, and the pellet was resuspended in 50 mL ofnormal saline. The pellet was resuspended in saline to yield a finalconcentration of 10 mg cytarabine per mL of suspension.

A laser diffraction particle size analyzer (Horiba Instruments, Irvine,Calif.) was used to determine particle size. The average length-weightedmean diameter of the resulting MVL particles was in the range from 12-16μm.

The use of different non-hydrohalic acids as release-modifying agentshad marked influence on the rate of cytarabine release from the MVLincubated in human plasma. The percent of cytarabine retained in the MVLafter incubation at 37° C. in human plasma for the different acids ismeasured as a function of time of incubation. The half-life of drugrelease, calculated assuming a single-exponential, is given in TABLE 2.The data in TABLE 2 are the mean and standard deviation from threeexperiments.

                  TABLE 2                                                         ______________________________________                                        Acid          Half Life in Days for Release of Cytarabine                     ______________________________________                                        Perchloric Acid                                                                             37.2 ± 8.0                                                   Nitric Acid   54.5 ± 5.7                                                   Phosphoric Acid                                                                             6.5 ± 0.2                                                    Formic Acid   5.6 ± 0.2                                                    Trichloroacetic Acid                                                                        5.5 ± 0.6                                                    Acetic Acid   4.8 ± 0.5                                                    Trifluoroacetic Acid                                                                        3.4 ± 0.4                                                    Sulfuric Acid 1.6 ± 0.5                                                    ______________________________________                                    

The nature of the release-rate modifying non-hydrohalic acid used toprepare the multivesicular liposomes had a profound effect on therelease rates of cytarabine in human plasma. Use of monoprotic inorganicacids, namely, nitric acid, and perchloric acid, resulted in the slowestrelease rate for cytarabine. Diprotic and triprotic acids, i.e.,sulfuric acid and phosphoric acid, resulted in fast release rates. Theorganic acids, formic acid, acetic acid, trifluoroacetic acid andtrichloroacetic acid, also resulted in fast release rates. Thus, adesired release rate can be achieved by selecting an appropriate acid asillustrated herein.

EXAMPLE 2

This example demonstrates that the rate of release of leuprolide fromMVL into an in vitro medium can be controlled by varying the acid.

Step 1) In a clean conical Teflon tube, 2 mL of a solution containing78.88 μmoles of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 16.65 μmolesof 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 118.8 μmoles ofcholesterol, 14.6 μmoles of triolein in chloroform were placed (thelipid component). The lipids were purchased from Avanti Chemical Company(Alabaster, Ala.).

Step 2) Two mL of first aqueous component, leuprolide (10 mg/nL)dissolved 0.1 M phosphoric acid, or ascorbic acid, or 0.2 M citric acid,or glucuronic acid, were added into the above Teflon tube containinglipid component.

Step 3) For making the water-in-oil emulsion, a homogenizer(AutoHomoMixer, Model M, Tokushu Kika, Osaka, Japan) was used by mixingfor 7 minutes at a speed of 10,000 rpm.

Step 4) For making the chloroform spherules suspended in water, 20 mL ofa solution containing 4 wt % glucose and 40 mM lysine was added to thewater-in-oil emulsion, and then mixed for 2 minutes at a speed of 2000rpm to form the chloroform spherules.

Step 5) The chloroform spherule suspension in the glass cylinder waspoured into the bottom of a 1000 mL Erlenmeyer flask containing 30 mL ofwater, 4 wt % glucose, and 40 mM free-base lysine. A stream of nitrogengas was passed at a flow-rate of 50 cu ft/hr over the suspension in theflask to evaporate chloroform over 20 minutes at 37° C. The MVL werethen isolated by centrifugation at 600 X g for 10 minutes.

The half life values in days for the plasma release were 15.8±8.4,4.7±1.5, 6.0±1.5, and 3.0±0.2, for phosphoric acid, ascorbic acid,citric acid, and glucuronic acid, respectively.

EXAMPLE 3

This example demonstrates that a mixture of zwitterionic amphipathiclipids and a neutral lipid can be used for producing the MVLcompositions with an acid used in the process.

The procedure for the preparation of MVL was the same as in EXAMPLE 1,with the following exceptions.

For Step 1, into a clean glass cylinder (2.5 cm inner diameter×10.0 cmheight) were placed 5 mL of a solution containing 13.20 μmoles of1,2-dioleoyl-sn-glycero-3-phosphocholine, 2.79 μmoles of1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 19.88 μmoles ofcholesterol, and 2.48 μmoles of triolein in chloroform (the lipidcomponent).

In Step 2, 5 mL of the first aqueous component and cytarabine (20 mg/mL)dissolved in 0.136 M sulfuric acid were added into the above glasscylinder containing the lipid component.

The half life value for in vitro release was 3.0±1.6 days.

EXAMPLE 4

This is an example for the antibacterial agent, amikacin, encapsulatedinto MVL in the presence of an non-hydrohalic acid.

The procedure for the preparation of MVL was the same as in EXAMPLE 1,with the following exceptions.

For Step 1, into a clean glass cylinder (2.5 cm inner diameter×10.0 cmheight) were placed 5 mL of a solution containing 13.20 μmoles of1,2-dioleoyl-sn-glycero-3-phosphocholine, 2.79 μmoles of1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 19.88 μmoles ofcholesterol, 2.48 μmoles of triolein in chloroform (the lipidcomponent).

In Step 2, 5 mL of the first aqueous component and amikacin (20 mg/mL)dissolved in 0.136 M sulfuric acid were added into the above glasscylinder containing the lipid component.

The half life value in plasma was 16.6±2.1 days.

Thus, the present disclosure provides "depot" preparations of wideapplication and uses in which biologically active substances areencapsulated in relatively large amounts, provide sustained exposure ordelivery at therapeutic concentrations of these substances for optimalresults, and the release rate of the substance is controlled by varyingthe nature of the acid used in the formulation.

For a given biologically active substance, one skilled in the art willbe able to choose an acid to produce an MVL composition with a desiredrelease rate of the encapsulated biologically active substance.

The present invention, therefore, is well suited and adapted to attainthe ends and objects and has the advantages and features mentioned aswell as others inherent therein.

While presently preferred embodiments of the invention have been givenfor the purpose of disclosure, it should be understood that variousmodifications can be made without departing from the spirit and scope ofthe invention. Accordingly, the following claims are intended to beinterpreted to embrace all such modifications.

What is claimed is:
 1. A multivesicular liposome having multiplenon-concentric chambers with internal membranes distributed as a networkthroughout, produced by a method comprising the steps of:(a) forming awater-in-oil emulsion from two immiscible components, the two immisciblecomponents being:1) a lipid component comprising at least one organicsolvent, at least one amphipathic lipid, and at least one neutral lipidlacking a hydrophilic head group, and 2) a first aqueous component; saidwater-in-oil emulsion further comprising:3) non-hydrohalic acid in aconcentration range from about 0.1 mM to about 0.5 M, wherein theconcentration is selected to provide controlled release of thebiologically active substance in 4) from the liposome, and 4) at leastone biologically active substance; said non-hydrohalic acid and saidbiologically active substance being independently incorporated into thelipid component, the first aqueous component, or both; (b) mixing thewater-in-oil emulsion containing the non-hydrohalic acid with a secondaqueous component to form solvent spherules; and thereafter (c) removingthe organic solvent from the solvent spherules to form multivesicularliposomes.
 2. The liposome of claim 1, wherein the acid is selected fromthe group consisting of sulfuric acid, phosphoric acid, and acetic acid,and combinations thereof and wherein the controlled release is atphysiologic conditions.
 3. The liposome of claim 1, wherein the acid isselected from the group consisting of nitric, formic, sulfuric,phosphoric, acetic, glucuronic, citric, and combinations thereof.
 4. Theliposome of claim 1, wherein the biologically active agent is selectedfrom the group consisting of an antitumor agent, an anaesthetic, ananalgesic, an antimicrobial agent, a hormone, an antiasthmatic agent, acardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, animmunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, aradionuclide, a radio contrast agent, a nucleic acid, a protein, aherbicide, a pesticide, and suitable combinations thereof.
 5. Theliposome of claim 1, wherein the biologically active substance iscytarabine.
 6. The liposome of claim 1, wherein the biologically activesubstance is amikacin.
 7. The liposome of claim 1, wherein thebiologically active substance is hydromorphone.
 8. The liposome of claim1, wherein the biologically active substance is leuprolide.
 9. Theliposome of claim 1, wherein the biologically active substance isinsulin.
 10. The liposome of claim 1, wherein the biologically activesubstance is interleukin-2.
 11. The liposome of claim 1, wherein thebiologically active substance is insulin-like growth factor-1.
 12. Theliposome of claim 1, wherein the biologically active substance is aninterferon.
 13. The liposome of claim 1, wherein the biologically activesubstance is granulocyte colony stimulating factor (G-CSF).
 14. Theliposome of claim 1, wherein the biologically active substance is tumornecrosis factor.
 15. The liposome of claim 1, wherein the biologicallyactive substance is tumor growth factor alpha.
 16. The liposome of claim1, wherein the biologically active substance is tumor growth factorbeta.
 17. The liposome of claim 1, wherein the biologically activesubstance is morphine.
 18. The liposome of claim 1, wherein thecontrolled release of the biologically active substance is sufficient toameliorate a disease following administration of the liposome to aliving mammal.
 19. The liposome of claim 1, wherein the biologicallyactive substance is selected from the group consisting of herbicides andpesticides.
 20. The liposome of claim 1, wherein the amphipathic lipidis provided in admixture with cholesterol, plant sterols, orcombinations thereof.
 21. The liposome of claim 1, wherein theamphipathic lipid is a zwitterionic lipid.
 22. The liposome of claim 1,wherein the amphipathic lipid is an anionic lipid.
 23. The liposome ofclaim 1, wherein the amphipathic lipid is a mixture of a zwitterioniclipid and an anionic lipid.
 24. The liposome of claim 1, wherein theamphipathic lipid is a mixture of a zwitterionic lipid and a cationiclipid.
 25. The liposome of claims 1, 20, 22, and 23, wherein thezwitterionic lipid is selected from the group consisting ofphosphatidylcholines, phosphatidylethanolamines, sphingomyelins,lysophosphatidylcholines, lysophosphatidylethanolamines, andcombinations thereof.
 26. The liposome of claims 1, 21, and 22, whereinthe anionic lipid is selected from the group consisting ofphosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidicacids, cardiolipins, and combinations thereof.
 27. Theliposome of claims 1 and 23, wherein the cationic lipid is selected fromthe group consisting of diacyl trimethylammonium propanes, diacyldimethylammonium propanes, stearylamine, and combinations thereof. 28.The liposome of claim 1, wherein the neutral lipid is selected from thegroup consisting of triglycerides, diglycerides, ethylene glycols, andcombinations thereof.
 29. The liposome of claim 1, wherein the organicsolvent is selected from the group consisting of ethers, hydrocarbons,halogenated hydrocarbons, halogenated ethers, esters, and combinationsthereof.
 30. The liposome of claim 1, wherein the emulsification of thetwo immiscible components is carried out using a method selected fromthe group consisting of mechanical agitation, ultrasonic energyagitation, and nozzle atomization.
 31. The liposome of claim 1, whereinthe formation of the solvent spherules is carried out using a methodselected from the group consisting of mechanical agitation, ultrasonicenergy agitation, and nozzle atomization.
 32. The liposome of claim 1,wherein the removal of the organic solvent is by a method selected fromthe group consisting of sparging, rotary evaporation, passing gas overthe solvent spherule suspension, solvent selective filtration, andcombinations thereof.
 33. The liposome of claim 1, wherein theconcentration of the organic solvent is in the range from about 3.98 mMto about 15 mM, the concentration of the amphipathic lipid is in therange from about 3.2 mM to about 47.77 mM, and the concentration of theneutral lipid is in the range from about 0.5 mM to about 7.3 mM.
 34. Theliposome of claim 33, wherein the amphipathic lipid is a combination of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in a concentration fromabout 2.64 mM to about 39.44 mM and1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) in a concentrationfrom about 0.56 to about 8.33 mM.
 35. The liposome of claim 1, whereinthe non-hydrohalic acid is selected from the group consisting of nitricacid, glucuronic acid, citric acid, formic acid, acetic acid, sulfuricacid, phosphoric acid, and combinations thereof.